ABSTRACT The ongoing HIV pandemic has resulted in ~1.2 million infections in the US and ~37 million infections worldwide. In spite of considerable progress in HIV/AIDS research, anti-retroviral therapy (ART) remains the only treatment option for HIV-1 infection. While ART has been highly effective in controlling the virus and making HIV infection a manageable disease, the drugs used in the ART regimen are expensive, cause side effects, and face viral resistance. Thus, there is an urgent need for continued development of drugs against novel cellular and viral targets. HIV-1 capsid protein (CA) is an important viral therapeutic target that is currently clinically unexploited. Recently, Gilead developing a highly potent CA-targeting inhibitor as next generation of antivirals. However, successful transition of these CA-specific inhibitors to the clinic require clear understanding of the anti-viral mechanisms and the delineating CA?s role in HIV-1 infection. Our studies are designed to understand the mechanism by which HIV-1 CA controls viral integration. HIV enters the target cell by fusion of the viral membrane with the cellular membrane, releasing the viral capsid core into the cytoplasm of the target cell. Functional studies of HIV-1 variants indicate that the proper assembly, morphology, and stability of the capsid core are all essential for HIV-1 infectivity. While it is well established that HIV-1 CA facilitates reverse transcription, recent data shows that CA is also a key determinant of the ability of HIV-1 to enter the nucleus of the target cell. In particular, CA is genetically and functionally implicated in nuclear entry of the reverse transcribed provirus, by mediating interactions with cellular factors. A key knowledge gap is the role of CA in viral integration-a critical post-nuclear entry step of HIV-1 infection. We hypothesize that HIV-1 CA protein directly influences viral DNA integration. To test this hypothesis we propose three specific aims: Aim 1. To define the effects of capsid stability on HIV-1 preintegration complex (PIC) activity and viral DNA integrity. Aim 2. To determine whether integration activity is dependent on CA levels in the PICs. Aim 3. To determine the role of known capsid-binding host proteins in defining PIC-associated CA levels and PIC activity. To test a direct link between HIV-1 CA and viral DNA integration, we have developed a novel model by coupling a biochemical approach that quantitatively measures PIC-associated integration activity to the use of the CA-specific inhibitor as probes. In addition, we have assembled a multidisciplinary team with expertise in PIC biochemistry (Dash), capsid biology (Aiken) and retroviral integration (Engelman). Therefore, the proposed studies will generate new knowledge on the mechanism by which CA regulates HIV-1 integration and define the antiviral effects of CA-inhibitors to facilitate the development of novel CA-based anti-viral therapies (a High-Priority HIV/AIDS research area of the NIH).